Polymicrobial biofilms of ocular bacteria and fungi on ex vivo human corneas

Microbes residing in biofilms confer several fold higher antimicrobial resistances than their planktonic counterparts. Compared to monomicrobial biofilms, polymicrobial biofilms involving multiple bacteria, multiple fungi or both are more dominant in nature. Paradoxically, polymicrobial biofilms are less studied. In this study, ocular isolates of Staphylococcus aureus, S. epidermidis and Candida albicans, the etiological agents of several ocular infections, were used to demonstrate their potential to form mono- and polymicrobial biofilms both in vitro and on human cadaveric corneas. Quantitative (crystal violet and XTT methods) and qualitative (confocal and scanning electron microscopy) methods demonstrated that they form polymicrobial biofilms. The extent of biofilm formation was dependent on whether bacteria and fungi were incubated simultaneously or added to a preformed biofilm. Additionally, the polymicrobial biofilms exhibited increased resistance to different antimicrobials compared to planktonic cells. When the MBECs of different antibacterial and antifungal agents were monitored it was observed that the MBECs in the polymicrobial biofilms was either identical or decreased compared to the monomicrobial biofilms. The results are relevant in planning treatment strategies for the eye. This study demonstrates that ocular bacteria and fungi form polymicrobial biofilms and exhibit increase in antimicrobial resistance compared to the planktonic cells.

Biofilm formation in ocular bacteria and fungi by the tissue culture plate method using crystal violet method. Biofilm formation was monitored in ocular isolates of S. aureus (L-1054-2019(2)), S. epidermidis (L-1058-2019(2)) and C. albicans (L-391-2015) by the tissue culture plate method (TCP) using crystal violet (CV) as described earlier 6,31 . In the CV method an overnight culture in YPD medium [(bacteriological peptone (20%), glucose(20%) and yeast extract (20%)] was diluted 10,000 times (v/v) and then 100 µl of the suspension (10 4 cells/ml) was incubated in a 96 well plate containing 100 µl of YPD medium at 37 °C for 24 h and 48 h. After incubation, the YPD medium was decanted, the planktonic cells discarded, and the cells that adhered to the wells were washed twice with 200 µl of phosphate-buffered saline (PBS), pH 7.4 (1× PBS contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 and 1.8 mM KH 2 PO 4 ), and plates air dried at room temperature. The bacterial cells that had adhered to the wells were stained using 0.1% aqueous crystal violet (Sigma Chemical Co., St. Louis, MO, USA). Excess crystal violet was discarded, and each well was washed twice with 200 µl of PBS and dried at RT. CV associated with the bacteria was extracted with 200 µl of absolute ethanol and quantified using a Spectrophotometer [SpectraMax M3, with a cuvette adaptor (Molecular Devices, San Jose, CA, USA)] set at 595 nm. Wells without cells served as the control (OD was < 0.1 at 595 nm) and the OD value was deducted from the "high-biofilm formers" (OD > 0.3 at 595 nm) and "low-biofilm formers" (OD < 0.3 at 595 nm) 32,33 . S. aureus ATCC25922 (positive for biofilm formation) and E. coli ATCC25923 (negative for biofilm formation) served as a positive and negative controls respectively for biofilm formation. The experiment was performed with three replicates.
Biofilm formation in ocular bacteria and fungi by the tissue culture plate method using XTT. In the XTT [2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (Sigma, USA) method 7,37 , cultures in YPD medium were diluted 10,000 times with YPD and then 100 µl of this suspension (10 4 cells/ml) was incubated in YPD in a 96 well plate for 24 h and 48 h. The media was then decanted, each well washed twice using 200 µl of autoclaved milliQ water and allowed to air dry for 30 min.The washed cells were stained in the dark with XTT by adding 200 µl of XTT solution [147 µl of PBS and 50.5 µl of XTT (1 mg/ ml, Sigma Chemical Co., St. Louis, MO, USA) and 2.5 µl of Menadione (0.4 mM, Sigma Chemical Co., St. Louis, MO, USA)] and incubated in the dark at 37 °C for 3 h. From each well, 100 µl was then transferred to a fresh 96 well plate and biofilm formation was quantified using a SpectraMax M3, microplate reader (Molecular Devices, CA, USA) 7,37 . Media without cells served as a negative control (OD was < 0.1 at 595 nm) and S. aureus ATCC 25922 (positive for biofilm formation) and E. coli ATCC 25923 (negative for biofilm formation) served as a negative and positive control respectively for biofilm formation with OD values of < 0.3 and > 0.3 respectively were considered as low-biofilm formers and high-biofilm formers.

Polymicrobial biofilm by simultaneous incubation of bacteria and fungi.
In polymicrobial biofilm formation more than one microorganism is monitored for biofilm formation. C. albicans along with either S. aureus or S. epidermidis were co-incubated in YPD media at 37 °C for 24 and 48 h at a final volume of 100 µl Biofilm thickness in monomicrobial and polymicrobial biofilms on human cadaveric corneas using confocal laser scanning microscopy. Monomicrobial and polymicrobial biofilms of ocular bacteria and the fungus were set up on on human cadaveric corneas as in the CV and XTT methods 34 and used for localisation of extracellular polymeric substance (EPS) and for the determination of the thickness of the biofilm by Confocal Laser Scanning Microscopy (CLSM) using dual staining 8 . After the incubation period of 24 and 48 h in RPMI medium, the human cadaveric corneas were washed with autoclaved distilled water and fixed with 250 µl of formaldehyde (4%) for 3 h. Fixed biofilms were then washed twice with autoclaved distilled water as above and stained for 30 min with 200 µl of 1.67 µM Syto9 (Invitrogen, Carlsbad, CA, USA), a nuclear fluorescent dye that stains DNA of viable cells and emits green color. After staining with Syto 9, biofilms were stained in the dark with 0.025% Calcofluor white M2R (Sigma Chemical Co., St. Louis, MO, USA) for 30 min. This dye binds to β-linked polysaccharides and fluoresces under long-wave UV light and biofilm could be visualized (blue) using confocal microscopy (Carl Zeiss LSM 880, Jena, Germany) 37 . The Argon Laser was excited at 450-490 nm for Syto9 and 363-nm using a 455/30 band-pass filter for Calcofluor white and a 20× objective was used set at Zoom 2. The thickness of the biofilm at each time point was measured across the biofilm and values are reported as Z axis, average ± standard deviation in µm. The data was analysed statistically using unpaired t-test. p value of < 0.05 was considered significantly different.
Visualisation of monomicrobial and polymicrobial biofilms on human cadaveric cornea using scanning electron microscopy. The procedure is identical to that described earlier 37 . Human cadaveric cornea which did not meet the stringent quality required for transplantation were obtained from The Ramayamma International Eye Bank (RIEB), LVPEI, Hyderabad, India. All corneas were obtained following procedures approved by the institutional review board for the protection of human subjects. Corneas were received in MK medium containing gentamicin 31 . These corneas were thoroughly washed with PBS prior to use for biofilm formation. The donor cornea with its epithelial surface facing upward was immersed in an antibiotic free RPMI (Roswell Park Memorial Institute) media 31,35 containing 10% fetal calf serum, 5 μg/ml insulin and 10 ng/ml epidermal growth factor and incubated for 24 h at 37 °C in a 5% CO 2 incubator to remove the residual antibiotics. Corneas from the antibiotic free RPMI medium were washed with PBS and a sterile steel scalpel was used to create three horizontal and vertical cuts 31,35 . Subsequently, the bacterial and fungal inoculum prepared from an overnight culture grown in YPD broth was diluted 10,000 times with YPD broth and centrifuged at 12,000 rpm (Eppendorf USA, Framingham, MA, USA, model no: 5430) for 5 min at room temperature (25 °C) and the pellet washed with 200 μl of autoclaved distilled water and centrifuged. The final pellet was suspended in 100 μl of RPMI and was gently transferred onto the surface of the corneas and incubated for 24 or 48 h at 37 °C in a CO 2 incubator (5% CO 2 in air). After the incubation period, the corneas were processed for SEM to visualize biofilms on the cornea. For this purpose,the corneas were washed with PBS, fixed with 2.5% glutaraldehyde (Himedia-Secunderabad, India) and washed again prior to dehydration through graded ethanol (10,25,50,70,90 and 100% for 20 min each) and finally air dried overnight. Biofilms on the corneas were sputtered with gold for 60 s using a high vacuum evaporator (SC7620 PALARON Sputter Coater, East Sussex, UK) and visualized using a scanning electron microcope (SEM) (Carl Zeiss-Model EVO 18, Carl Zeiss, Germany). The voltage used for acquiring the SEM images ranged between 5 and 20 kV 31 .
Antimicrobial susceptibility in planktonic phase. Several antimicrobials (antifungals and antibacterials) were evaluated for their antimicrobial activity as per Clinical and Laboratory Standards Institute (CLSI) (drivingitproductivity.com/2021/10/28/clsi-guidelines-for-antimicrobial-susceptibility-testing/). An overnight bacterial suspension in YPD broth was adjusted to 0.5 McFarland units, diluted 100-fold and 100 µl of the suspension was added to each well of a 96 well polystyrene plate (Nunclon™, Thermo Scientific, Roskilde, Denmark) containing 100 µl of an antifungal/antibacterial agent of a known concentration. Minimum inhibitory concentration (MIC/MBEC) for each antimicrobial agent was determined in three replicates according to CLSI-M07-A10 guidelines as described earlier [6][7][8]31 .
Antibiotic susceptibility in monomicrobial biofilms. Inhibitory effects of antimicrobials on monomicrobial biofilms was performed as described earlier [6][7][8]31 . Briefly, an overnight culture of bacterium or fungus in YPD medium was diluted (10 4 cells/ml) and was allowed to form a biofilm at 37 °C for 24 h as described above. The YPD medium was decanted, the wells washed twice with PBS to remove the planktonic cells and the required concentration of the antimicrobial agent was added. After incubation for additional 24 h the wells were washed with 200 µl of PBS to remove the planktonic cells and the plates were then processed for monitoring the effect of the antimicrobial agent by the XTT method [6][7][8]31 . Inoculums without the addition of the compound served as a negative control. All experiments were performed in triplicate. www.nature.com/scientificreports/ Antibiotic susceptibility in polymicrobial biofilms. Bacteria plus fungi were incubated simultaneously in YPD to form a biofilm for 24 h after which the planktonic cells were discarded, the biofilm washed twice with PBS and then the antibacterial or antifungal agent was added for an additional 24 h. Subsequently, the biofilms were washed, and the effect of the antimicrobial agent was evaluated by the XTT method 31 . In a separate experiment either the bacterium or the fungi were allowed to form a biofilm for 24 h after which the other was added and additionally incubated for another 24 h. At the end of the 48 h incubation period the biofilms were washed and then the antibacterial or antifungal agent was added for an additional 24 h. Subsequently, the biofilms were washed, and the effect of the antimicrobial agent was evaluated by the XTT method as described 31 . Inoculums without the addition of the compound served as a negative control. All experiments were performed in triplicate.

Statistical analysis.
All experiments were performed in triplicates and standard deviation was calculated.
Wherever applicable all comparisons were evaluated using unpaired t test for proportions and homogeneity and a p ≤ 0.05 was considered significant.
Ethical approval. All experiments were performed in accordance with relevant guidelines and regulations of the L V Prasad Eye Institute, Hyderabad, India and the experimental protocols were approved by the Institutional Review board and the Institutional ethics committee of the L V Prasad Eye Institute, Hyderabad, India (LEC-BHR-P-04-21-623). Additionally, informed consent was obtained from the legal guardians of all the donors. All the donors were adults and in the age group of 22-35 years.

Results
In this study both the XTT and CV methods were used for quantification of the biofilms. The XTT method measures cell viability 31 whereas the CV method measures cell wall material and biofilm matrix 31 .  Table 1). Increase was more pronounced in S. aureus plus C. albicans polymicrobial biofilm at 48 h compared to 24 h biofilm formation when the OD doubled (Supplementary Table 1). Further, when C. albicans was added to preformed biofilms of S. epidermidis significant increase in XTT positive (metabolically active cells) was consistently observed compared to the monomicrobial biofilm of S. epidermidis at 48 h ( Fig. 1B; Supplementary Table 1). Thus, polymicrobial biofilms are more pronounced compared to the monomicrobial biofilms with respect to metabolically active cells in the biofilm (also see supplementary Table 1).

Quantification of polymicrobial biofilms of bacteria and fungi by the XTT method. The quan-
Quantification of polymicrobial biofilms of bacteria and fungi by the tissue culture plate method using crystal violet. In the TCP method using crystal violet (CV) only the polymicrobial biofilm wherein C. albicans biofilm was preformed and then S. aureus was added, significant increase was observed only with the monomicrobial S. aureus biofilm ( Fig Staphylococcus epidemidis and C. albicans polymicrobial biofilm incubated simultaneously showed significant increase in biofilm formation compared to monobacterial biofilm at 48 h. Polymicrobial biofilm of C. albicans to which S. epidermidis was added also showed increase in biofilm formation compared to both monobacterial and monofungal biofilms ( Visualisation of monomicrobial and polymicrobial biofilms of S. aureus and C. albicans on human cadaveric cornea using scanning electron microscopy. Staphylococcus aureus and C. albicans formed monomicrobial biofilms by 24 h and multilayer clumping of cells was visible, and EPS was sparingly seen (Fig. 4a,b). In C. albicans at 24 h hyphae were also visible (Fig. 4b). When cultured together at 24 h both S. aureus and C. albicans formed polymicrobial mixed biofilms with S. aureus forming small clumps on the surface of C. albicans (Fig. 4c). Further when C. albicans was added to 24 h preformed biofilm of S. aureus polymicro- www.nature.com/scientificreports/ bial mixed biofilms were formed and clumping of both the bacteria and fungi was more intense, hyphae were more prominent, and EPS was clearly visible (Fig. 4d) compared to when the bacterium and fungi were cultured together to form the polymicrobial biofilm (Fig. 4c). By 48 h the monomicrobial biofilms of S. aureus and C. albicans formed denser clumps and produced more EPS compared to 24 h of monomicrobial biofilms (Fig. 4e,f). Further, when S. aureus and C. albicans were grown simultaneously for 48 h the polymicrobial biofilms exhibited clumping and the S. aureus were totally enclosed in EPS (Fig. 4g). But, when C. albicans monomicrobial biofilm was preformed for 24 h and then S. aureus was added the resulting polymicrobial mixed biofilms exhibited intense clumping of both the bacteria and fungi, hyphae were more prominent and EPS was clearly visible (Fig. 4h).
Visulisation of monomicrobial and polymicrobial biofilms of S. epidermidis and C. albicans on human cadaveric cornea using scanning electron microscopy. Staphylococcus epidermidis like S. aureus formed monomicrobial biofilms which appeared as multi-layered clumps of cells and EPS was visible (Fig. 5a). The biofilm by 48 h appeared as huge column of multilayered biofilm covered with EPS ( Fig. 5e compared with Fig. 5a). But when S. epidermidis was simultaneously induced to form biofilm along with C. albicans, the biofilm was not very prominent at 24 h ( Fig. 5c) but it was more dense and only a few bacteria were visible at 48 h (Fig. 5g). Further when S. epidermidis was cultured for 24 h and then C. albicans was added, clumping of the two microbes was observed separately and a few colonised the surface of C. albicans hyphae and yeast forms (Fig. 5d). The polymicrobial biofilm between the bacterium and fungi was more prominent when C. albicans was allowed to form biofilm for 24 h and then S. epidermidis was added (Fig. 5h).
Antibiotic susceptibility of S. aureus in monomicrobial (S. aureus) and polymicrobial (S. aureus plus C. albicans) biofilms with planktonic cells of S. aureus. The concentration at which the XTT method indicated < 0.3 OD 490 nm was taken to be the MBEC, since at this concentration none of the cells were viable. The MBEC of S. aureus in the biofilm phase was increased several fold (> 2 fold) compared to the planktonic cells for all the 18 different antibiotics that were screened (Supplementary Table 3; Fig. 6A). Amikacin, ceftriaxone, chloramphenicol and ofloxacin at 12 ug/ml were the most effective in the planktonic phase which increased to several fold (32-512 μg/ml) in the biofilm phase at 48 h of biofilm formation (Supplementary Table 3; Fig. 6A). The MBECs of all the 18 antibiotics increased in the polymicrobial biofilm phase irrespective     Table 4). In the reverse experiment wherein C. albicans formed a biofilm for 24 h and then S. epidermidis was added the results indicated that the MBEC to most of the antibiotics (12) decreased whereas MBEC of the remaining 6 antibiotics remained unchanged (chloramphenicol, azithromycin, metronidazole, clindamycin, lincomycin and monocycline) compared to the biofilm of S. epidermidis at 24 h and 48 h (Supplementary Table 4).

Susceptibility of C. albicans to antifungal agents in a monomicrobial (C. albicans) and polymicrobial (C. albicans plus S. aureus or S. epidermidis) biofilm. Candida albicans in the biofilm phase
was less susceptible compared to the planktonic cells to the 6 antifungal agents tested and was most susceptible to caspofungin (MBEC, 20 μg/ml) at 48 h of biofilm growth (Supplementary Table 5). Polymicrobial mixed biofilm of S. aureus and C. albicans when incubated together (simultaneously) was less susceptible compared to the planktonic cells of C. albicans and the MBECs were identical to the MBECs of monomicrobial biofilm of C. albicans at 48 h (Fig. 8A, Supplementary Table 5). Further when C. albicans biofilm was performed for 24 h and then S. aureus cells were added the mixed polymicrobial biofilm after 24 h showed further decrease in MBEC to 4 of the 6 antibiotics whereas susceptibility to caspofungin and fluconazole were similar compared to the mixed simultaneously formed biofilm and monospecies biofilm of C. albicans at 48 h (Supplementary Table 5). In reverse experiments when the biofilm was preformed by S. aureus and then C. albicans was added the MBEC decreased with respect to Fluconazole compared to when C. albicans biofilm was preformed ( Fig. 8B; Supplementary Table 5) and MBEC of 5 antibiotics decreased (except capsofungin) with respect to simultaneously formed biofilm at 24 h and monospecies biofilm of C. albicans at 48 h. Polymicrobial mixed biofilm of S. epidermidis and C. albicans when incubated together (simultaneously) was less susceptible compared to the planktonic cells of C. albicans (Fig. 8A, Supplementary Table 5). MBECs of these simultaneously mixed biofilms exhibited decrease in MBECs for all antibiotics compared to MBECs of monomicrobial biofilm of C. albicans at 48 h (Fig. 8B, Supplementary Table 5). In similar experiments, when C. albicans biofilm was preformed to which S. epidermidis was added the MBECs were identical to that seen when S. aureus was added to C. albicans biofilm (Fig. 8B, Supplementary Table 5). But when the biofilm of S. epidermidis was performed and then C. albicans was added the mixed biofilm response was identical to the simultaneously formed biofilm of C. albicans and S. epidermidis (Fig. 8B, Supplementary Table 5). MBECs for all antibiotics were decreased with respective to monospecies C. albicans biofilm at 48 h. But when biofilm of C. albicans was performed and S. epidermidis was added the MBECs were very similar except for caspofungin and fluconazole which showed increase in MBECs compared to the simultaneously formed biofilm of C. albicans and S. epidermidis (Fig. 8B, Supplementary Table 5).

Discussion
The ocular bacteria Staphylococcus aureus and S. epidermidis and the fungus C. albicans 7,32 have earlier been reported by us to form monomicrobial biofilms 7,31,32 . Additionally, it is demonstrated for the first time that these ocular bacteria along with the fungus form polymicrobial biofilms. One earlier study had indicated that nonclinical strains of S. aureus and C. albicans form a polymicrobial biofilm 36 . C. albicans, is conducive to polymicrobial biofilm formation with other bacteria such as Streptococcus mutans, Fusobacterium spp. and E. coli 37 . Mixed fungal-bacterial biofilms of C. albicans and E. coli or S. aureus were reported on endotracheal tubes and urinary catheters, and Aspergillus fumigatus and Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients 38,39 . Ocular isolates of S. aureus, S. epidermidis and C. albicans are known to cause several eye diseases. For instance, S. aureus causes dacryocystitis, conjunctivitis, keratitis, cellulitis, corneal ulcers, blebitis, and endophthalmitis 40,41 , S. epidermidis causes blepharitis and suppurative keratitis and C. albicans causes endophthalmitis or choroiditis 42 . In this study, XTT method and the TCP spectrophotometric method consistently demonstrated that C. albicans formed polymicrobial mixed biofilms when incubated together (simultaneously) with either S. aureus or S. epidermidis (Figs. 1, 2; also see Supplementary Tables 1, 2) or when the bacterium or the fungus were sequentially added to one another after 24 h (Figs. 1, 2; Supplementary Tables 1, 2). The minor discrepancy in the results between the XTT and TCP methods could be attributed to the differences in the two methods. The XTT method reflecting the number of viable and metabolically active cells 43 whereas in the TCP www.nature.com/scientificreports/ method crystal violet binds cells as well as matrix components 44 . Several studies have already demonstrated the potential of ocular isolates to form monobacterial biofilms on indwelling ocular medical devices 26,31 (see "Introduction"). But, we are not aware of any studies on polymicrobial biofilms involving ocular bacteria and fungi as in this study. Polymicrobial biofilms involving either species of the same genus or species from different kingdoms (such as bacteria and fungi) are probably more dominant in nature 45 . Such polymicrobial biofilms are clinically relevant since they are associated with several ocular infections and infections of the lung, inner ear, urinary tract, oral cavity, wounds, teeth and those that dwell on devices 46,47 . Further, the dual species involved in the formation of polymicrobial biofilms varied depending on the infection 48,49 . The commonly encountered microorganisms in polymicrobial biofilms were S. aureus-Pseudomonas aeruginosa 50 , S. aureus-C. albicans 51 , S. aureus-C. tropicalis and C. tropicalis-S. marcescens 52 , Staphylococcus xylosus-S. aureus 53 etc. It was observed that ocular isolates of C. albicans, S. aureus and S. epidermidis formed polymicrobial mixed biofilms irrespective of whether the bacteria and fungus were added simultaneously onto the substratum, or the bacterium was added to the preformed fungal biofilm or vice versa. In the latter case, the occurrence of mixed biofilms was indicative that the ocular bacteria and the fungus could penetrate preformed biofilms as reported earlier in mixed biofilms of S. aureus and P. aeruginosa 50 .  (Figs. 4, 5). In the ocular isolates the monomicrobial biofilms at 48 h showed increased clumping of cells and excessive of EPS (Fig. 4a,e, 5a,e) compared to the polymicrobial mixed biofilms (Figs. 4c,d,g,h, 5c,d,g,h) implying that in the dual species the interaction between the taxa may be influencing the biofilm process. Further, when C. albicans monomicrobial biofilm was preformed for 24 h and then S. aureus was added or vice versa polymicrobial mixed biofilms were denser, hyphae were more prominent, and EPS was clearly visible (Fig. 4d,h).
Confocal microscopy studies indicated that the thickness of the polymicrobial mixed biofilms (S. aureus plus C. albicans and S. epidermidis plus C. albicans) increased compared to the monomicrobial biofilms ( Fig. 3; also see Supplementary Figs. 1, 2) when the bacteria and fungi were incubated simultaneously to form biofilm. Interestingly it was also observed that when the fungus biofilm was preformed and then either of the bacteria were added to the biofilm the thickness increased. But biofilm thickness did not exhibit significant increase in thickness when the biofilm was preformed by bacteria to which the fungus was added (Fig. 3). The reason for this is not clear but it could imply that the preformed fungal biofilm is not conducive to the establishment of the polymicrobial biofilm by bacteria. Earlier we had shown that theses ocular isolates of S. aureus, S. epidermidis The effect of the antimicrobial agent was evaluated by the XTT method as described. The coloured bars indicate the following: red square, C. albicans in the planktonic phase (24 h); blue square, C. albicans in the biofilm phase (24 h); green square, S. epidermidis plus C. albicans simultaneously incubated to form biofilm (24 h); purple square, C. albicans biofilm preformed for 24 h and then S. epidermidis planktonic cells were added; brown square, S. epidermidis biofilm preformed for 24 h and then C. albicans planktonic cells were added; orange square, S. aureus plus C. albicans simultaneously incubated to form biofilm (24 h); light blue square, C. albicans biofilm preformed for 24 h and then S. aureus planktonic cells were added; pink square, S. aureus biofilm preformed for 24 h and then C. albicans planktonic cells were added. Experiments were performed in triplicates. www.nature.com/scientificreports/ and C. albicans formed monomicrobial biofilms which increased in thickness with incubation period 7,31 . Several other studies also indicated that biofilm thickness increases with incubation period 54 .

Scientific
Interactions between the microbes in a polymicrobial biofilm have been implicated in disease progression, in causing an inflammatory state, in inducing collateral damage in the host, in increasing tolerance to biotic stresses due to host and predatory microorganisms and in exhibiting drug resistance and tolerance to antibiotics 10,47,55,56 . Our results confirm that the monomicrobial biofilms exhibited several-fold more resistance to all the antimicrobials tested compared to planktonic cells (Figs. 6, 7, 8; Supplementary Tables 3-5) confirming earlier studies on ocular S. aureus 31,41,57 , S. epidermidis 31,58 and C albicans 7 . Earlier studies had also indicated that polymicrobial mixed biofilms were more resistant to antimicrobials compared to the monomicrobial biofilms biofilms 36,55,[59][60][61] . For example, a polymicrobial biofilm of C. albicans and S. aureus was more resistant to vancomycin and daptomycin than as a monoculture 36 . Staphylococcus epidermidis, has also been shown to protect C. albicans from the action of the antifungal drugs fluconazole and amphotericin B in polymicrobial biofilms 62 . In this study, when the resistance of the polymicrobial mixed biofilms were compared to monomicrobial biofilms the MBEC values either remained unchanged or decreased (Fig. 6, 7, 8; also see Supplementary Table 3) except in one case when S. aureus biofilm was performed and then C. albicans was added the MBEC of Ampicillin increased (Supplementary Table 3; Fig. 6). Increase in resistance of polymicrobial mixed biofilms to antimicrobials compared to the monomicrobial biofilms has been attributed to poor antibiotic penetration, nutrient limitation, slow growth, stress, formation of persister cells and extracellular biofilm matrix formation 36,62,63 . Interaction between the taxa in a polymicrobial biofilm has also been implicated in enhanced tolerance to antibiotics 64 . For instance in polymicrobial biofilm C. albicans enhanced the resistance of S. aureus 61,65,66 to daptomycin and vancomycin 36 . In cystic fibrosis (CF) mixed polymicrobial biofilm with P. aeruginosa, and Inquilinus limosus or with Dolosigranulum pigrum increase the tolerance to most antibiotics 67 . In this study the resistance of the polymicrobial mixed biofilms to several antibiotics was either identical or decreased compared to that of the monomicrobial biofilm. This observation contradicts earlier studies which had also indicated that polymicrobial biofilms are more challenging to treat since they are more resistant to antimicrobial treatment than the corresponding single-species biofilms 21,22 and corresponding planktonic cells 23 . Identical MBECs to antibiotics in the polymicrobial biofilm would imply that the observed increased thickness of the biofilm in the polymicrobial biofilm ( Fig. 3; also see Supplementary Figs. 1, 2) may not be influencing the resistance to antimicrobials. Further, the resistance to antimicrobials decreased in the polymicrobial mixed biofilms compared to the monomicrobial biofilm in many instances (Figs. 6, 7, 8; also see Supplementary  implying that the biotic components (bacteria and fungi) within the biofilm were interacting and making them more sensitive to the drugs 50 . Trizna et al. 50 had earlier demonstrated that in S. aureus and P. aeruginosa dual species biofilms a tenfold increase in susceptibility to ciprofloxacin and aminoglycosides (gentamicin or amikacin), was observed compared to monobacterial biofilms. The results imply that strategies used to hack monobacterial biofilms should be equally efficient in targeting microbes in a polymicrobial mixed biofilm.
To the best of our knowledge this is the first study demonstrating that ocular bacteria and fungi possess the potential to form polymicrobial mixed biofilms which exhibit increased resistance to both antibacterial and antifungal agents compared to planktonic cells.
Relevance of the study. The above results are of relevance to ocular infection treatment. In an ocular clinic handling ocular surface infections the organism that first appears in culture from an ocular sample is the target of treatment. But this may not be the best approach in case of polymicrobial infections since fungal infections are normally detected after a week on culturing, whereas bacterial infections appear within 48 h. Thus, bacteria become the first targets of medication. It is good to start the treatment to target the first detected organism, but one should also look for other organisms which may appear with time, and they also need to be treated. If a polymicrobial infection is not considered or is missed, the outcome may be adversely affected 4 .

Conclusions
1. Antibiotic and antifungal susceptibility studies confirmed that S. aureus, S. epidermidis and C. albicans in the monomicrobial biofilm phase were several fold more resistant to antimicrobial agents compared to the planktonic phase. 2. Ocular isolates in polymicrobial mixed biofilm phase are also more resistant to antimicrobials compared to the planktonic cells. 3. Ocular isolates in the polymicrobial mixed biofilm phase most often showed no change or decreased resistance to antimicrobials compared to the monomicrobial biofilm phase organisms. 4. Considering that the chosen ocular bacteria and fungus are the etiological agents of several ocular diseases the studies would be very relevant in planning treatment strategies for the eye.

Limitations.
1. The study does not address the cellular-basis of polymicrobial biofilm formation? For instance, when the bacteria or fungi are added to an already formed monomicrobial biofilm how do they attach to the monomicrobial biofilm? Further, it is not clear whether they first attach to the substratum and then to the biofilm or vice versa? 2. Need to study the expression of genes associated with biofilm formation in monomicrobial and polymicrobial biofilms?